The Study On The Antioxidative Effect Of Astaxanthin On The Stored Suspended Red Blood Cells Membrane

Suspended red blood cell is one kind of blood product which is the most widely used.Red blood cell infusion has become an important clinical treatment method to increase oxygen transport capacity of blood and improve the hypoxia state of tissue.But with the increasing of storage time,red blood cells will emerge a series of changes on metabolism and structure.for example,gradual degradation of various components in cells,reduction of oxygen carrying capacity and deformation capacity,these changes are referred to as "storage damage".Clinical studies show that: The transfusion of suspended red blood cells which emerge "storage damaged" may lead to post transfusion complications and increase the mortality of patients.Red blood cell membrane is the key to maintain the normal structure and function of red blood cell.There are many important functional proteins,structural proteins and active proteins in the membrane.The character of red blood cell membrane is an important factor for whether red blood cell can survive in vivo after transfusions.The research team has confirmed that: with the storage time extension of red blood cell membrane,the structure would be destroyed,red blood cell would expand and volume would increase.So,the change of structure and function of red blood cell membrane is an important part for "storage damage".Oxidative stress is one of the main mechanisms which lead to the "storage damage: red blood cells are rich in hemoglobin,during storage,hemoglobin is constantly oxidized automatically and produce a large number of reactive oxygen species?ROS?.ROS can oxidize the lipids,active proteins and structural proteins in the red blood cell membrane,change the structure and function of cell membrane,even cause cell death.So far,astaxanthin?ASTA?is one of the powerful antioxidants found in nature.The hydroxyl group,conjugated double bond and ketone group of ASTA can provide electrons to free radicals,quench singlet oxygen and finish the peroxidation reaction quickly.Research found:ASTA could reduce the oxidative stress level of nervous system diseases,cardiovascular system diseases and ischemia-reperfusion injury,no obvious side effects was found.In this study,ASTA was used to treat the suspended red blood cells,to observe the effect of ASTA on ROS content in cell,cell ultrastructure,membrane peroxidation damage degree and the activities of Superoxide dismutase?SOD?,Na⁺-K⁺-ATPase and Ca²⁺-ATPase.Objective: In this study,the antioxidant ASTA was added into the maintenance solution of suspended red blood cells.On the 7th and 28 th day of storage,the level of oxidative stress,the degree of membrane peroxidation damage,the cell ultrastructure,the activities of SOD,Na⁺-K⁺-ATPase and Ca²⁺-ATPase were observed,to explore whether the antioxidant ASTA can improve the degree of cell membrane peroxidation injury,increase the activity of SOD,Na⁺-K⁺-ATPase and Ca²⁺-ATPase,decrease the ultrastructural changes of the suspended red blood cells by reducing the oxidative stress level in suspended red blood cells.Methods: 1 The preparation and groups of suspended red blood cellsThe blood of 6 healthy volunteers were collectd,400 ml per person.The blood were made into suspended red blood cells and regarded as experimental specimen after the HBV,HCV,HIV,TP dection were negative.One bag of suspended red blood cells was prepared and slightly reversed to make the red blood cells uniformly distribute.The blood was sub-packed in two empty blood bags by sterile interface machine and randomly divided into control group and ASTA group.In the sterile operation table,The ASTA dissolved by DMSO was added into the blood bag of ASTA group by disposable syringe.Then the blood bags was gently reversed to make ASTA uniformly distributed in blood bag.The final concentration of ASTA was 10mmol/L.Only the same amount of DMSO solvent was added into the blood bag of control group.The red blood cells of control group and ASTA group were sub-packed into two empty blood bags by sterile interface machine again.At last,all the suspended red blood cells were stored in refrigerator at 2 ?-6 ?.The extracellular fluid and red blood cells were collected on the 7 and 28 day of storage respectively to detect the related indexes.2 The detecting indexes and methods 2.1 The ultrastructural changes of suspended red blood cells1ml of suspended red blood cells was centrifugated for 5 minutes at 1000 rpm and 4 ?,then the supernatant was discarded.After washing 3 times by Normal saline,the blood was fixed for 10 min by 1% glutaraldehyde at room temperature,then the supernatant was removed.The three steaming water was add into the red blood cells and stood for 5mins at room temperature to wash the suspended red blood cells,the supernatant was removed.Then,the red blood cells were repeatly washed two times.After dehydration by gradient ethanol,vacuum drying,gold plating,the ultrastructural changes of suspended red blood cells were observed by S-3500 N Hitachi scanning electron microscope.And 4 fields were randomly selected to count the number of the biconcave disc-shaped red blood cells and spinous red blood cells,calculate the proportion average value?%?.2.2 The detection of ROS content in suspended red blood cellsAccording to the operating instructions of ROS test kit,the DCFH-DA?diluted with 1:1000 normal saline?added into to the suspended red blood cells washed by normal saline,then incubated in 37 ?cell incubator.The suspended red blood cells were lysed with Ripa cell lysate of equal volume after the reaction was finished.The fluorescence value in the lysed product was detected by Fluorescent enzyme labeling instrument.At the same time,the hemoglobin content in lysate of suspended red blood cells was detected by hemoglobin test kit.The ratio of fluorescence value to hemoglobin content in lysate was used to express the ROS content in suspended red blood cells.2.3 The detection of extracellular FHb of suspended red blood cells2 ml of stored suspended red blood cells was collected,and centrifugated for 10 minutes at 3000 rpm and 4 ?,the supernatant was collected for FHb detection The content of extracellular FHb of suspended red blood cells was determined by chemical colorimetry.2.4 The extraction of suspended red blood cells membrane1ml of suspended red blood cells was centrifugated for 5 minutes at 500 rpm and 4 ?,then the supernatant was discarded.3 times volume of Normal saline was added into the red blood cells sedimentation and gently upside down to wash the red blood cells.Then the suspended red blood cells were centrifugated for 5 minutes at 500 rpm and 4 ?,the supernatant was discarded,washing three times like above to get clean red blood cells.40 times volume of sodium phosphate buffer?5 mmol/L,PH8.0?was add into red blood cells and shook violently for 15 minutes to make it hemolysis.The hemolytic product of red blood cells were centrifugated for 40 minutes at 20000 rpm,4 ? and discarded the upper liquid.The white precipitated part was the suspended red blood cells membrane.After the suspended red blood cells membrane was washed twice with sodium phosphate buffer of equal volume,the sedimentation of red blood cells membrane was collected again and diluted with normal saline,then stored in-70 refrigerator for detection.2.5 The detection of MDA content in suspended red blood cells membraneThe content of MDA in suspended red blood cells membrane was determined by thiobarbituric acid colorimetry.The assay process was carried out according to the operating instructions of the kit.2.6 The detection of SOD activity in suspended red blood cells membraneThe SOD activity in suspended red blood cells membrane was measured by xanthine oxidase method.The assay process was carried out according to the operating instructions of the kit.2.7 The detection of Na⁺-K⁺-ATPase activity in suspended red blood cells membraneThe Na⁺-K⁺-ATPase activity in suspended red blood cells membrane was measured by colorimetry.The assay process was carried out according to the operating instructions of the kit.2.8 The detection of Ca²⁺-ATPase activity in suspended red blood cells membraneThe Ca²⁺-ATPase activity in suspended red blood cells membrane was measured by colorimetry.The assay process was carried out according to the operating instructions of the kit.2.9 The correlation analysis of SOD activity,Na⁺-K⁺-ATPase activity,Ca²⁺-ATPase activity in suspended red blood cells membrane and MDA content in cell membrane and ROS content in red blood cells.The correlation analysis of SOD activity,Na⁺-K⁺-ATPase activity,Ca²⁺-ATPase activity in suspended red blood cells membrane and MDA content in cell membrane and ROS content in red blood cells were analyzed by Pearson correlation analysis.Results:1.The ultrastructural changes of suspended red blood cellsUnde the scanning electron microscope : When stored for 7 days,most of the red blood cells of the control group were biconcave disc shape,but the concave became shallow,red cell swelled,rounded bulge appeared on the edge of red blood cells,a few cells appeared spinous protuberance?thorn?,the concave of suspended red blood cells in ASTA group was obvious,the swelling was lighter than that of the control group,rounded bulge also appeared on the edge of red blood cells,individual cell were spinous.The number of spinous protuberance red blood cells was less than that of the control group?P < 0.01?.When stored for 28 days,a large number of suspended red blood cells in control group were spinous,the protuberance of thorn was obvious.The number of spinous protuberance red blood cells of ASTA group was significantly less than that of control group?P < 0.05?,some biconcave disc-shaped red blood cells could be seen.2.The ROS content in suspended red blood cells?fluorescence intensity/ mg Hb?When stored for 7 days,the ROS content in suspended red blood cells of control group was 204.26±28.69,the ROS content of ASTA group was 148.13±5.49,the ROS content in suspended red blood cells of ASTA group was significantly lower than that of control group?p<0.01?;when stored for 28 days,the ROS content in suspended red blood cells of control group was 274.98±27.2,the ROS content of ASTA group was 183.63±16.08,the ROS content in suspended red blood cells of ASTA group was significantly lower than that of control group?P<0.01?3.The extracellular FHb content of suspended red blood cellsWhen stored for 7 days,the extracellular FHb content of suspended red blood cells of control group was 52.77±5.35 mg/L,the extracellular FHb content of ASTA group was 36.13±5.59 mg/L,the extracellular FHb content of suspended red blood cells of ASTA group was significantly lower than that of control group?P<0.01?;when stored for 28 days,the extracellular FHb content of suspended red blood cells of control group was 148.82±5.81 the extracellular FHb content of ASTA group was 96.13±11.85 mg/L,the extracellular FHb content of suspended red blood cells of ASTA group was significantly lower than that of control group?P<0.01?.4.The MDA content in suspended red blood cells membraneWhen stored for 7 days,the MDA content in suspended red blood cells membrane of control group was 25.61±3.88 mmol/g,the MDA content of ASTA group was 10.78±1.68 mmol/g,the MDA content in suspended red blood cells membrane of ASTA group was significantly lower than that of control group?P<0.01?;when stored for 28 days,the MDA content in suspended red blood cells membrane of control group was 52.65±7.38mmol/g,the MDA content of ASTA group was 31.09±4.59mmol/g,the MDA content in suspended red blood cells membrane of ASTA group was significantly lower than that of control group?P<0.01?.5.The SOD activity in suspended red blood cells membraneWhen stored for 7 days,the SOD activity in suspended red blood cells membrane of control group was 0.62±0.08U/mg pro,the SOD activity of ASTA group was 1.02±0.16 U/mg pro,the SOD activity in suspended red blood cells membrane of ASTA group was significantly higher than that of control group?P<0.01?:when stored for 28 days,the SOD activity in suspended red blood cells membrane of control group was 0.32±0.05 U/mg pro,the SOD activity of ASTA group was 0.65±0.10 U/mg pro,the SOD activity in suspended red blood cells membrane of ASTA group was significantly higher than that of control group?P<0.01?.6.The Na⁺-K⁺-ATPase activity in suspended red blood cells membraneWhen stored for 7 days,the Na⁺-K⁺-ATPase activity in suspended red blood cells membrane of control group was 3.65±0.64 mmol /mg pro·h,the Na⁺-K⁺-ATPase activity of ASTA group was 4.56±0.68 mmol /mg pro·h,the Na⁺-K⁺-ATPase activity in suspended red blood cells membrane of ASTA group was significantly higher than that of control group?P<0.05?;when stored for 28 days,the Na⁺-K⁺-ATPase activity in suspended red blood cells membrane of control group was 2.51±0.36 mmol /mg pro·h,the Na⁺-K⁺-ATPase activity of ASTA group was 3.35±0.45 mmol /mg pro·h,the Na⁺-K⁺-ATPase activity in suspended red blood cells membrane of ASTA group was significantly higher than that of control group?P<0.01?.7.The Ca²⁺-ATPase activity in suspended red blood cells membraneWhen stored for 7 days,the Ca²⁺-ATPase activity in suspended red blood cells membrane of control group was 6.25±0.79 mmol /mg pro·h,the Ca²⁺-ATPase activity of ASTA group was 7.78±0.99 mmol /mg pro·h,the Ca²⁺-ATPase activity in suspended red blood cells membrane of ASTA group was significantly higher than that of control group?P<0.05?;when stored for 28 days,the Ca²⁺-ATPase activity in suspended red blood cells membrane of control group was 4.39±0.58 mmol /mg pro·h,the Ca²⁺-ATPase activity of ASTA group was 5.92±0.78 mmol /mg pro·h,the Ca²⁺-ATPase activity in suspended red blood cells membrane of ASTA group was significantly higher than that of control group?P<0.01?.8.The correlation of SOD activity,Na⁺-K⁺-ATPase activity,Ca²⁺-ATPase activity in suspended red blood cells membrane and MDA content in cell membrane and ROS content in red blood cells.There were negative correlation between SOD activity,Na⁺-K⁺-ATPase activity,Ca²⁺-ATPase activity in suspended red blood cells membrane and MDA content in cell membrane and ROS content in red blood cells.Conclusions:1.ASTA can reduce the oxidative stress level of stored suspended red blood cells,decrease the peroxidation damage degree of cell membrane and the ultrastructure change of cell.2.ASTA can increase the activities of SOD,Na⁺-K⁺-ATPase and Ca²⁺-ATPase in the suspended red blood cells membrane,delay the decreasing of important enzyme protein activity in cell membrane.